Processes for nucleic acid based assays are well known and have been implemented in a variety of formats. One of the more common processes conducted in such a nucleic acid based assay is a nucleic acid amplification process. Generally, a nucleic acid amplification reaction is first carried out to completion, and then a nucleic acid probe is used to determine the presence or absence of an amplified nucleic acid sequence of interest. This type of assay is referred to as an end point assay.
One problem with end point assays is that the amplified nucleic acid (amplicons) from the amplification reaction must be physically transferred to the subsequent probe assay. Because of the transfer, the potential exists for contaminating the laboratory environment with the amplicons. In addition, the general risk of misidentifying a given sample or confusing it with other samples increases each time that a physical transfer of the sample takes place.
Thus, there have been previous proposals for self-contained test units that are capable of carrying out an integrated nucleic acid amplification and nucleic acid probe assay on a liquid biological sample while the sample remains confined within the test unit. For example, U.S. Pat. No. 5,229,297, to Paul N. Schnipelsky et al., describes a cuvette for DNA amplification and detection which comprises a plurality of flexible compartments for containing a sample, amplifying reagents and detection reagents, together with passageways connecting the sample and reagent compartments with a detection site and waste compartment. A roller is used to squeeze or compress the sample and reagent compartments in a desired sequence, thereby forcing the sample and detection reagents through the passageways to the detection site and waste compartment. Temporary seals are used to isolate the sample and reagent compartments from the passageways until sufficient pressure is generated by the roller. Although this arrangement is advantageous in that the sample remains within the cuvette during amplification and detection, the need for a roller to break the temporary seals and cause the various fluids to flow between compartments introduces undesirable complexity and makes it difficult to automate the amplification and assay procedure.
Furthermore, in U.S. Pat. No. 5,639,428, an improved test unit for carrying out integrated nucleic acid amplifications and nucleic acid probe based detection assays is disclosed. In the improved test unit, the flow of sample and reagent liquids is controlled by centrifugal force applied by a rotating apparatus, thereby avoiding the need for rollers and other complex mechanisms. While this represents a substantial improvement over the arrangement disclosed in U.S. Pat. No. 5,229,297, the need to provide for controlled fluid movement within the test unit still exists and renders the test unit somewhat more complex than might be desired.
In addition to the end point assays discussed previously, homogeneous real time methods of nucleic acid detection assay also exist. Homogeneous real time methods do not require the physical transfer of the amplified material to a separate assay site, but rather function simultaneously with the amplification reaction, thus, detection can occur in real time. Examples of known homogeneous real time methods include fluorescence polarization, fluorescence energy transfer and light absorbance.
As with the end point assays, there have also been previous proposals for self-contained test units for homogeneous assay methods. For example, U.S. Pat. No. 5,236,827 and its counterpart European Patent No. 0 347 771, describe a device with a fluorogenic substrate for conducting an assay in which an enzyme rate-of-reaction profile is determined to identify microorganisms.
European Patent Application No. 0 640 828 describes an instrument for monitoring multiple nucleic acid amplifications simultaneously. The instrument includes a thermal cycler and a sensor for detecting emitted light from multiple amplifications simultaneously.
U.S. Pat. No. 5,219,762 describes a device and method for measuring the product of an enzymatic reaction wherein the enzyme acts on a target analyte to produce a detectable and measurable enzymatic byproduct.
European Patent No. 0 298 669 describes methods for performing nucleic acid reactions and manipulations in reaction vessels with reagents in a dried state.
Also, in copending U.S. patent application Ser. No. 08/878,096, filed Jun. 18, 1997, a device and method for a homogeneous fluorescence polarization assay is described. This device contains all of the reagents necessary for both a nucleic acid amplification reaction and a nucleic acid probe based assay in dried form, such that all such reagents will be rehydrated by a liquid biological sample at essentially the same time. The device is configured as a flat card in order to minimize the amount of sample in each sample cell of the device, and thereby allow for preheating of the device and rapid equilibration of the temperature of added liquid sample to the temperature of the preheated device (i.e. a "hot start" of the method). However, the rehydration of all dried nucleic acid amplification reagents and all dried nucleic acid probe assay reagents at essentially the same time has been found to cause an unreproducible fluorescence detection signal. This unreproducible signal is believed to be due to variable rehydration of the fluorescently labeled dried nucleic acid probe, and causes interference with the desired fluorescence signal, which interference cannot be factored out, because of the unreproducible nature of the interfering signal. Also, the small volumes used in this device make detection of amplicons from some samples more difficult, and render the device not easily usable by those in the clinical diagnostics field.
In view of the foregoing, a need exists in the art for a kit and method for carrying out an homogeneous nucleic acid amplification and real time nucleic acid probe detection assay with minimal complexity, and which is capable of yielding a consistent, reliable fluorescence detection signal.